A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice

Abstract

Objective: The objective of this study was to demonstrate that islet neogenesis-associated protein (INGAP) peptide, a pentadecapeptide containing the biologically active portion of native INGAP, increases functional beta-cell mass in normal animals and can be used therapeutically to reverse hyperglycemia in streptozotocin-induced diabetes.

Summary background data: INGAP, a 175 amino acid pancreatic acinar cell protein, has been suggested to be implicated in beta-cell mass expansion.

Methods: In the first part of this study, normoglycemic hamsters were administered either 500 microg INGAP peptide (n = 30) or saline (n = 20) intraperitoneally daily and sacrificed after 10 or 30 days of treatment. Blood glucose and insulin levels were measured, and a histologic and morphometric analysis of the pancreas was performed to determine the effect of INGAP peptide on the endocrine pancreas. In the second part of the study, 6- to 8-week-old C57BL/6J mice (n = 8) were administered multiple low doses of the beta-cell toxin streptozotocin (STZ) inducing insulitis and hyperglycemia. The mice were then injected with INGAP peptide (n = 4) or saline (n = 4) for 39 days and sacrificed at 48 days. Two additional groups of diabetic mice were administered either a peptide composed of a scrambled sequence of amino acids from INGAP peptide (n = 5) or exendin-4 (n = 5), an incretin that has been associated with amelioration of hyperglycemia.

Results: Islet cell neogenesis was stimulated in INGAP-treated hamsters by 10 days. At 30 days, the foci of new endocrine cells had the appearance of mature islets. There was a 75% increase in islet number, with normal circulating levels of blood glucose and insulin. Administration of INGAP peptide to diabetic mice reversed the diabetic state in all animals, and this was associated with increased expression of PDX-1 in duct cells and islet cell neogenesis with a reduction of insulitis in the new islets. Diabetic mice treated with exendin-4 or a scrambled INGAP peptide did not revert from hyperglycemia.

Conclusion: Because there is a deficiency of beta-cell mass in both type-1 and type-2 diabetes, INGAP peptide stimulation of fully functional neoislet differentiation may provide a novel approach for diabetes therapy.

FIGURE 1. Histologic features of the pancreas after administration of INGAP peptide. A: β-cell neogenesis (arrow) in the wall of a small intralobular duct after 10 days of INGAP peptide treatment. To the left is a normal islet. Magnification: 200×. B: An area of new islet formation outside its putative duct of origin (arrowheads) at 30 days. The red stain identifies insulin protein and was visualized by immunocytochemistry. Magnification: 200×.

FIGURE 2. Change in the islet population induced by INGAP peptide. Normal Syrian Golden hamsters were treated with saline (□, n = 10) or INGAP peptide (▪, n = 15) for 10 or 30 days. Islet number (per mm²) and islet size (μm²) as determined by computer-assisted morphometric analysis. A: INGAP peptide-treated animals exhibited a significant increase in islet number (*P <0.01). B: This was the result of new islet formation as reflected by the shift in the distribution of islet size (**P <0.05) to small islets.

FIGURE 3. A: β-cell mass of normal Syrian golden hamsters treated with saline (□) or INGAP peptide (▪) for either 10 days or 30 days as determined by computer-assisted morphometric analysis. B: Increase in pancreatic insulin content after 10 days of INGAP peptide treatment. *P <0.025, **P <0.01.

FIGURE 4. Induction of β-cell neogenesis by INGAP peptide. Normal Syrian golden hamsters were treated with saline (□) or INGAP peptide (▪) for either 10 days or 30 days. A: Duct-associated β-cell mass and B: extra-islet acinar-associated β-cell mass as determined using a computer-assisted morphometric system. *P <0.05.

FIGURE 5. Increase in pancreatic endocrine cell mass with escalating INGAP peptide dose. Normal CD-1 mice treated with saline (□) or INGAP peptide (▪) at 50, 500, or 2500 μg/d for 31 days. β-cell mass was determined using a computer-assisted morphometric system. *P <0.05 versus saline.

FIGURE 6. Reduction in blood glucose by treatment with INGAP peptide. C57BL/6J mice were rendered diabetic using a regimen of multiple injections of low-dose streptozotocin. The animals were then randomized to receive daily treatment with either saline (□) or 500 μg/day INGAP peptide (▪) for 39 days. *P <0.025.

FIGURE 7. Histologic characteristics of the pancreas of streptozotocin-treated C57BL/6J mice with and without INGAP peptide treatment. A: Pancreas of an INGAP peptide-treated animal showing an area of islet cell neogenesis, observed as endocrine-like cells budding from an adjacent intralobular ductule. Magnification: 200×. B: A normal-appearing islet in a pancreas of an animal treated with INGAP peptide. Magnification: 220×. C: The pancreas of a saline-treated animal showing a necrotic islet with inflammatory cell infiltration characteristic of insulitis. Magnification: 350×. D: The pancreas of a normal age-matched non-treated control mouse showing the histologic appearance of a normal islet for comparison. H and E stained sections. Magnification: 350×.

FIGURE 8. Expression of PDX-1 and insulin in the pancreas of streptozotocin-treated C57BL/6J mice administered either saline (□) or INGAP peptide (▪) for 39 days. A: PDX-1 expression form an INGAP-treated animal (magnification: 850×) and B: quantified by computer-assisted morphometric analysis. C: Focal area of duct-associated islet neogenesis in an INGAP-treated animal stained for both PDX-1 and insulin protein (magnification: 820×). *P <0.01.

FIGURE 9. Immunocytochemistry of islet cell hormones in the pancreas of streptozotocin-treated diabetic C57BL/6J mice administered INGAP peptide. A: An islet associated with a segment of duct epithelium (to the right) is stained for insulin (black) and B: for both glucagon and somatostatin demonstrating a normal presence and distribution of islet cell hormones (magnification: 300×).